For the structural analysis of high molecular compounds, such as sugar chains or peptides, an ion trap mass spectrometer including a MALDI (matrix-assisted laser desorption ionization) ion source and a three-dimensional quadrupole ion trap has been widely used. There are two types of systems for performing a mass spectrometry of various kinds of ions temporarily held in an ion trap; one type uses the mass-separating function of the ion trap itself, while the other type ejects ions from the ion trap and detects those ions after separating them according to their masses by a time-of-flight mass spectrometer provided outside the ion trap. In the following description, the two types of systems are collectively referred to as an ion trap mass spectrometer.
A generally used analytical technique for high molecular compounds by an ion trap mass spectrometer is as follows.
After various kinds of ions obtained by ionizing a target compound by a MALDI method are captured in an ion trap, an ion-selecting operation is performed in such a manner that a kind of ion having a specific mass-to-charge ratio m/z is selectively retained as a precursor ion within the ion trap, while the other kinds of ions are ejected to the outside of the ion trap. Subsequently, a collision-induced dissociation (CID) gas is introduced into the ion trap, and the precursor ion is excited to make it collide with the CID gas and promote its dissociation. If the target structure cannot be adequately dissociated by a single CID operation, the selection of the precursor ion and the CID operation may be repeated a plurality of times. As a result of the CID operation thus performed one or more times for ions originating from the compound to be analyzed, a number of finely fragmented product ions are obtained, which are subsequently subjected to an ion-detecting process with a mass scan to obtain an MSn spectrum. By analyzing this MSn spectrum, the structure of the target compound is deduced.
In general, in an ion trap mass spectrometer, an operation called the “cooling” is performed in order to gather captured ions around the center of the capturing space of the ion trap for the purpose of improving the detection sensitivity and the mass-resolving power. That is to say, a cooling gas, which is an inert gas such as helium (He), is introduced into the ion trap and the captured ions are made to come in contact with the cooling gas so as to lower the kinetic energy of the ions. The ions having the kinetic energy thus decreased are more easily affected by the capturing electric field. Therefore, they do not widely spread within the capturing space but are more likely to gather around the center of the capturing space. In the case of the previously described sequential processes for the mass spectrometry, the cooling is normally performed after the ions are introduced into the ion trap from outside. The cooling is also performed after a precursor ion is dissociated by the CID operation and the thereby produced product ions are captured by the capturing electric field.
It is often the case that a high molecular compound to be analyzed by the previously described ion trap mass spectrometer includes a modification or functional group that easily dissociates Typical examples of such modifications or functional groups include sialic acids, sulfate groups and phosphate groups. It is commonly known that, when a sugar chain to which sialic acid is bonded (which is a kind of acidic sugar), or a glycopeptide to which a sialic-acid-bonded sugar chain is added, is dissociated by a low-energy CID in an ion trap mass spectrometer using a MALDI ion source, the sialic acid is preferentially dissociated.
However, the dissociation of sialic acid easily occurs not only in the CID process; it can also easily occur due to an in-source decay or a collision with the cooling gas, as well as due to a post-source decay if a time-of-flight mass spectrometer is used. Therefore, peaks of ions produced by a partial or entire dissociation of sialic acids are also observed even in a normal mass spectrometry in which no CID operation is performed (see Non-Patent Document 1 or other documents). Thus, particularly in the case of an ion trap mass spectrometer using a MALDI ion source, there is the problem that, if a compound to which an easily dissociable modification like the aforementioned ones is bonded is contained in the unknown sample, both the peaks of ions from which the modification has been dissociated and the peaks of ions from which the modification has not been dissociated will appear in the mass spectrum, making it difficult to determine the assignment of the ion peaks.
Furthermore, in the case where the assignment of the peaks is determined based on the mass-to-charge-ratio difference between each pair of the peaks in a mass spectrum obtained by a mass spectrometry of a target compound to which the aforementioned modification is bonded, if there is a peak which is unrelated with the target compound and yet has a mass-to-charge ratio that accidentally coincides with that of the dissociated modification (e.g. an impurity peak or noise peak), the assignment of that peak will be incorrectly determined, making the identification of the target compound difficult or incorrect.
In the case where the identification or structural analysis of an N-linked glycopeptide is performed by using an ion trap mass spectrometer, the following problem also exists: An MS2 spectrum obtained for an N-linked glycopeptide has three characteristic peaks appearing at predetermined intervals of mass-to-charge ratio (which are hereinafter called the “triplet peaks”), which specifically includes a peptide ion resulting from complete dissociation of sugar, a 0.2X(83Da)-added peptide ion resulting from a cross-ring cleavage of the HexNAc sugar, and a HexNAc(203Da)-added peptide ion arranged in ascending order of mass-to-charge ratio. Therefore, the MS2 spectrum normally is initially analyzed for the neutral losses of the sugar to locate triplet peaks, after which an MS3 analysis with an ion corresponding to these peaks designated as the precursor ion is performed. Then, based on the thereby obtained MS3 spectrum, the peptide and the glycosylation site are identified.
In general, since the amount of ions detected in an MS2 analysis is smaller than in the normal mass spectrometry (MS1 analysis) in which no CID operation is performed, it is necessary to increase the number of signal accumulations so as to create an MS2 spectrum with adequate strength, which means that the MS2 analysis must be repeated an accordingly large number of times. As a result, a long period of time is required to identify a peptide, and in the case of an analysis of a trace amount of sample originating from a living body, the sample may possibly be exhausted in the middle of the analysis, making it impossible to identify a peptide. Furthermore, in the case where 2,5-dihydroxybenzoic acid (DHB), which is recognized to be suitable for the ionization of glycopeptides in the MALDI method, is used as the matrix, it is known that the matrix sublimates during an analysis in vacuum atmosphere, terminating the ionization of the sample (i.e. the peptide). Thus, it has been a major problem for an analysis of N-linked glycopeptides to shorten the period of time required for the identification of a peptide.